Kinetochores are proteinaceous machines that coordinate numerous events during chromosome segregation, including moving chromosomes, generating spindle checkpoint signals and correcting improper microtubule attachments. Kinetochores bind and regulate the plus-ends of microtubules to perform these cellular processes. This proposal dissects the mechanisms of end-on attachment using a structure-function analysis of key components in a combination of in vitro and in vivo assays. We will dissect the role of a microtubule binding activity on the Ndc80/Hec1 subunit of the Ndc80 complex, which is proposed to be an important microtubule interface. We will also dissect the function of a new player in kinetochore regulation. Cep57 is a protein that we have shown recently is required for end-on attachment. Cep57 plays a different role in the process than Ndc80 and is required for attachment even when Ndc80 is unaffected. Cep57 directly binds microtubules and a number of regulators of kinetochore dynamics, placing it at a critical interface between the proteins that bind microtubules and the proteins that regulate end-on attached microtubules. Finally, we will perform a structural analysis of the outer kinetochore plate to understand why so many proteins in the kinetochore directly contact the microtubule. Our data suggest that the kinetochore has enormous potential as a previously untapped target for anti-mitotic chemotherapy that could have a profound impact on the treatment of human disease. PUBLIC HEALTH REVELANCE: The missegregation of chromosomes during mitosis is a major source of genetic mutations in cancer. During mitosis, every chromosome assembles two large proteinaceous machines called kinetochores that drive the segregation of the replicated DNA strands to the two daughter cells. Kinetochore proteins are often mutated in cancers, and this machine has become an important new target for chemotherapeutics. The experiments in this proposal will elucidate the roles of important proteins in the kinetochore, with the long-term goal of understanding how this complex machine segregates chromosomes and how cancers change their proteins to evolve more rapidly.